title: Folate polyglutamylation eliminates dependence of activity on enzyme concentration in mitochondrial serine hydroxymethyltransferases from Arabidopsis thaliana.
abstract: The reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT) is the major one-carbon unit source for essential metabolic processes. The Arabidopsis thaliana genome encodes seven SHMT isozymes localized in mitochondria, plastids, nuclei, and the cytosol. Knowledge of the biochemical properties of each isozyme is central to understanding and manipulating one-carbon metabolism in plants. We heterologously expressed and purified three recombinant SHMTs from A. thaliana (AtSHMTs) putatively localized in mitochondria (two) and the cytosol (one). Their biochemical properties were characterized with respect to the impact of folate polyglutamylation on substrate saturation kinetics. The two mitochondrial AtSHMTs, but not the cytosolic one, had increased turnover rates at higher (>0.4ng/μL) enzyme concentrations in the presence of monoglutamylated folate substrates, but not in the presence of pentaglutamylated folate substrates. We found no experimental support for a change in oligomerization state over the range of enzyme concentration studied. Modeling of the enzyme structures presented features that may explain the activity differences between the mitochondrial and cytosolic isozymes.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164, USA.
authors.forename: Zhaoyang
authors.initials: Z
authors.lastname: Wei
authors.forename: Kehan
authors.initials: K
authors.lastname: Sun
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Sandoval
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Cross
authors.forename: Christine
authors.initials: C
authors.lastname: Gordon
authors.forename: ChulHee
authors.initials: C
authors.lastname: Kang
authors.forename: Sanja
authors.initials: S
authors.lastname: Roje
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D005492:Folic Acid / Q000378:metabolism*; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013763:Tetrahydrofolates / Q000378:metabolism
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; C097188:polyglutamine; D005492:Folic Acid; D012696:Glycine Hydroxymethyltransferase
keywords: Folate polyglutamylation; One-carbon metabolism; Serine hydroxymethyltransferase (SHMT); Tetrahydrofolate